Project description:Analysis of keratinocyte differentiation in presence or absence of fibroblast-derived JNK-dependent soluble factors in vitro. The hypothesis tested in the present study was that JNK-dependent fibroblast-derived soluble factors promote an efficient keratinocyte differentiation. Results provide important information about reduced expression of a subset of differentiation associated genes in keratinocytes in the absence of JNK-dependent fibroblast-derived soluble factors. In vitro transwell co-culture experiments were performed using jnk1-/-jnk2-/- or wildtype immortalized mouse embryonic fibroblasts (MEFs) and differentiating primary normal human epidermal keratinocytes (NHEK) over a time course of 6 days. Total RNA was obtained from NHEK prior to cultivation with MEFs (day 0) and every second day (days 2, 4, and 6) during co-culture. MEFs have been described previously in Sabapathy et al. (2004) Mol Cell 15:713-25.
Project description:Analysis of JNK-dependent fibroblast-derived soluble factors at gene expression level. The hypothesis tested in the present study was that loss of c-Jun N-terminal kinases 1 and 2, JNK1 and JNK2, in MEFs causes a strong alteration of the gene expression program coding for soluble factors, which promote an efficient keratinocyte differentiation. Results provide important information of the repertoire of fibroblast transcripts encoding secreted proteins, which is severely disarranged upon loss of JNK activity under the in vitro co-culture conditions applied. In vitro transwell co-culture experiments were performed using jnk1-/-jnk2-/- or wildtype immortalized mouse embryonic fibroblasts (MEFs) and differentiating primary normal human epidermal keratinocytes (NHEK) over a time course of 6 days. Every second day, fibroblast-loaded inserts were changed resulting in 3 triads (triads 1, 2, and 3). Total RNA was obtained from jnk1-/-jnk2-/- and wildtype immortalized mouse embryonic fibroblasts (MEFs) prior to co-cultivation (day 0) and of each triad 1, 2, or 3.
Project description:Analysis of cultured epidermal keratinocytes treated with interleukin-4 (IL-4) and interleukin-13 (IL-13). IL-4 and IL-13 are up-regulated in atopic dermatitis. Results provide insight into the role of IL-4 and IL-13 cytokines in the pathogenesis of atopic dermatitis. Analysis of epidermal keratinocytes transfected with dual oxidase 1 (DUOX1) siRNA knockdown before treatment with IL-4 and IL-13. DUOX1 is one of the NOX family members of NADPH oxidases whose primary function is ROS generation. Results provide insight into the role of the incraesed expression of DUOX1 in IL-4/IL-13-treated NHEK for IL4/IL13 signaling. IL-4 and IL-13 induced gene expression in human epidermal keratinocytes (NHEK) was measured at 48 hours. Gene expression in NHEK tranfected with 10 nM DUOX1 siRNA followed by treatment with 100 ng/ml IL-4 and 100 ng/ml IL-13 was measured at 48 hours. Three independent experiments were performed using different strains for each experiment.
Project description:Atopic dermatitis (AD) is a serious inflammatory skin disorder characterized by increased levels of proinflammatory cytokines that contribute to a vicious cycle of inflammation. While the in-flammatory recombinant human epidermal (RHE) models related to AD have been established, there is currently lack of comprehensive understanding. To reveal the alterations and identify potential hub genes in AD-related inflammation, related RHE models induced by inflammatory cocktail (polyinosinic-polycytidylic acid, TNF-α, IL-4 and IL-13) are constructed and analyzed through TMT-proteomic in combination with RNA-seq transcriptomic.
Project description:Examination of binding locations of Pax3 and Pax7 in primary myoblasts UCSC track hub available at: http://www.ogic.ca/projects/Soleimani_2012_Pax7_hub/hub.txt For details on viewing the track hub in the UCSC Genome Browser: http://altair.dartmouth.edu/ucsc/goldenPath/help/hgTrackHubHelp.html#View 3 Samples (Control, Pax7 ChIP, Pax3 ChIP)
Project description:The antagonistic actions of Polycomb and Trithorax are responsible for proper cell fate determination in mammalian tissues. In the epidermis, a self-renewing epithelium, previous work has shown that release from Polycomb repression only partially explains differentiation gene activation. We now show that Trithorax is also a key regulator of epidermal differentiation, not only through activation of genes repressed by Polycomb in progenitor cells, but also through activation of genes independent of regulation by Polycomb. The differentiation associated transcription factor GRHL3/GET1 recruits the ubiquitously expressed Trithorax complex to a subset of differentiation genes. Examination of WDR5 and GRHL3 binding in human differentited primary keratinocytes (NHEK). High calcium medium was added to NHEK cells at 50% confluency to induce differentiation. Cells were collected for ChIP 24 hours after addition of high calcium medium. ChIP with Wdr5 and Grhl3 antibodies, and an input control were sequenced.
Project description:Aristolochic acid nephropathy (AAN) is characterised by rapidly progressive tubulointerstitial nephritis culminating in end stage renal failure and urothelial malignancy. microRNAs (miRs) are small endogenous post-transcriptional regulators of gene expression implicated in numerous physiological and pathological processes. We aimed to characterise the mechanism of AA induced cell cycle arrest and its regulation by miRs. The microarray experiment was performed to identify differentially regulated microRNAs in human proximal tubulal epithelial cells treated with aristolochic acid (AA). Analysis or differential miR expression in human proximal tubular epithelial cell line (HK-2) treated with 5ug/ml aristolochic acid, control (n=3) vs aristolochic acid (n=3)
Project description:This SuperSeries is composed of the following subset Series: GSE25064: ChIP-Seq of Pax7 and Pax3 in myoblasts GSE32266: Mouse Myoblast Pax3, Pax7 overexpression and control Refer to individual Series
Project description:Advanced head and neck squamous cell carcinomas (HNSCC) are frequently drug resistant and have a mortality rate of 40%. We have previously shown that E2F7 may contribute to drug sensitivity. In the present study, we conducted an M-bM-^@M-^Somics screen to identify factors that were responsible for E2F7-dependent resistance to anthracyclines by HNSCC. We provide, in vitro, in vivo and patient data that identifies the existence of a novel E2F7/Sphingosine kinase 1/Sphingosine-1-phosphate/AKT axis that regulates sensitivity to anthracyclines in HNSCC. Specifically, we show that E2F7-dependent sensitivity to doxorubicin occurs via induction of the sphingosine kinase 1/AKT axis. We also show that pharmacological inhibition of Sphingosine kinase 1 or AKT sensitizes SCC cells to the cytotoxic actions of doxorubicin in vitro and in vivo. Combined, these findings highlight a novel mechanism through which SCC cells acquire resistance to anthracyclines and identify specific pharmacological combinations that could be used to treat SCC. Duplicate total RNA isolated from i) untreated, KJD cells, ii) 1M-BM-5M doxorubicin-treated KJD cells, iii) control plasmid (pcDNA3) transfected, untreated KJD cells, iv) control plasmid (pcDNA3) transfected, 1M-BM-5M doxorubicin-treated KJD cells, v) E2F7 expression plasmid transfected, untreated KJD cells, vi) E2F7 expression plasmid transfected, 1M-BM-5M doxorubicin-treated KJD cells, vii) untreated, SCC25 cells, viii) 1M-BM-5M doxorubicin-treated SCC25 cells, ix) control siRNA (GFPsiRNA) transfected, untreated SCC25 cells, x) control siRNA (GFPsiRNA) transfected, 1M-BM-5M doxorubicin-treated SCC25 cells, xi) E2F7siRNA transfected, untreated SCC25 cells, xii) E2F7siRNA transfected, 1M-BM-5M doxorubicin-treated SCC25 cells were hybridised to an RNA microarray to identify genes which are downstream of E2F7 and regulate chemosensitivity in SCC.
Project description:Cancer stem cells (CSCs) drive tumour spread and therapeutic resistance, and can undergo epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) to switch between epithelial and post-EMT sub-populations. Examining oral squamous cell carcinoma (OSCC), we now show that increased phenotypic plasticity, the ability to undergo EMT/MET, underlies increased CSC therapeutic resistance within both the epithelial and post-EMT sub-populations. The post-EMT CSCs that possess plasticity exhibit particularly enhanced therapeutic resistance and are defined by a CD44highEpCAMlow/-CD24+ cell surface marker profile. Treatment with TGFβ and retinoic acid (RA) enabled enrichment of this sub-population for therapeutic testing, through which the endoplasmic reticulum (ER) stressor and autophagy inhibitor Thapsigargin was shown to selectively target these cells. Demonstration of the link between phenotypic plasticity and therapeutic resistance, and development of an in vitro method for enrichment of a highly resistant CSC sub-population, provides an opportunity for the development of improved chemotherapeutic agents that can eliminate CSCs. The clonal derivative of the CA1 OSCC cell line termed 18 here (otherwise termed pEMT-P) was treated with 0.5ng/ml TGFbeta and/or 5uM RA for eight days, with media changed every second day. The LM OSCC cell line was FACS sorted to recover the CD44highEpCAMlowCD24+ sub-population, which was then treated with 0.5ng/ml TGFbeta and/or 5uM RA for eight days, with media changed every second day. The parental CA1 and LM cell lines are included as reference samples.